For therapeutic applications, immunoglobulins must be of very high product quality. This requires, amongst others, homogeneity in structural terms. Moreover, the production costs are strongly influenced by difficulties encountered during the production process. For example, difficulties to separate structural variants of the desired protein will necessitate complex and costly purification strategies. Lack of homogeneity will impact the economics of the production process and hence, the costs for the therapeutic overall. Lack of homogeneity may also increase undesired properties of the immunoglobulin, such as the immunogenicity.
Heterologous expression of biopharmaceuticals is common. The transformation and manipulation of well-studied hosts such as Escherichia coli or Saccharomyces cerevisiae draw on many genetic tools and strains and a plethora of research into regulatory responses and protein functions. Non-conventional yeasts, in particular Pichia pastoris, have found wide application in heterologous expression because they produce very high levels of heterologous proteins, secrete proteins, or exhibit more versatile substrate utilization. However, relatively few genetic tools, engineered strains and data on the biology of these organisms are available.
When over-expressing in a host a protein destined for secretion from the host into the culture medium, the secretion of the protein can be achieved by fusing the protein to a secretion signal sequence that directs the passage of the polypeptide through the endoplasmic reticulum and the Golgi apparatus and that is cleaved from the polypeptide by a signal peptidase before being packaged into secretory vesicles, which fuse with the plasma membrane to release the processed protein.
A secretion signal sequence that has been used to produce a variety of secreted proteins in yeast expression systems is the signal sequence of the prepro-polyprotein precursor of the S. cerevisiae mating pheromone alpha-factor (aMF secretion signal peptide; Kurjan and Herskowitz 1982, Cell 30: 933-943; Sasagawa et al. 2011, Plasmid 65(1): 65-69).
While the use of the aMF secretion signal peptide most often results in secretion of the proteins from the yeast strains, it has been observed that the proteins are not always correctly processed. For example, WO 2002/086101 describes that the expression in Hansenula polymorpha and Saccharomyces cerevisiae of HCV envelope proteins using the alpha-mating factor leader sequence of S. cerevisiae leads to several expression products with a different amino-terminus in addition to the main product. WO 2007/148345 describes that the expression in Pichia pastoris of recombinant Exendin-4 using the alpha factor signal sequence and propeptide results, in addition to the main product, in molecules that are clipped, lacking the first two amino acids from the N-terminus.
During secretion of the alpha mating factor signal sequence from S. cerevisiae, the signal peptide is removed by two processing enzymes. The enzyme Kex2 cleaves after a specific lysine-arginine, then Ste13 clips off the dipeptides EA and EA to yield the mature alpha mating factor (Fuller et al. 1988, Annu. Rev. Physiol. 50: 345-362). Esposito et al. 2005 (Prot. Expr. and Purif. 40: 424-428) observed that while the Pichia homolog of Kex2 could cleave at the expected site, the Ste13 homolog did not, resulting in N-terminal extension of amino acid residues EAEA derived from the secretion signal sequence. They stated that inefficient Ste13 processing of the alpha mating factor signal peptide in Pichia is common.
Ghosalkar et al. 2008 (Protein Expr. Purif. 60(2): 103-109) also describe that the expression in Pichia pastoris of human interferon-alpha 2b (IFN-alpha2b) using, amongst others, the full Saccharomyces cerevisiae MF-alpha factor prepro sequence resulted, in addition to the main product, in two molecules with a different N-terminus due to inefficient processing of the secretion signal. However, the alpha prepro sequence without the EAEA repeats (i.e. with only the Kex2 cleavage site) directed the secretion of maximum amount of IFN-alpha2b into the culture medium, with the same amino terminal sequence as the native protein.
In contrast to the difficulties described above, immunoglobulin single variable domains can be readily expressed in P. pastoris. Immunoglobulin single variable domains are characterized by formation of the antigen binding site by a single variable domain, which does not require interaction with a further domain (e.g. in the form of VH/VL interaction) for antigen recognition. Production of Nanobodies, as one specific example of an immunoglobulin single variable domain, has been extensively described e.g. in WO 94/25591 and is known to result in good quality product. Moreover, as outlined in WO 2010/125187, material produced in P. pastoris is characterized by equal functionality as compared to E. coli produced material. Up to now the problem of incompletely processed product being present in the final product has not yet been observed for immunoglobulin single variable domains.